Immunoassay for homocysteine

ABSTRACT

The invention relates to a method for assaying homocysteine in a sample such as blood, plasma or urine, which comprises the steps of contacting the sample with a homocysteine converting enzyme and at least one substrate for the enzyme other than homocysteine, and without chromatographic separation, assessing a non-labelled analyte selected from a homocysteine co-substrate and the homocysteine conversion products of the enzymic conversion of homocysteine by said enzyme.

The present invention relates to an assay for homocysteine in clinicalsamples.

Homocysteine is an intermediary amino acid produced when methionine ismetabolised to cysteine. Generally, homocysteine produced in the body israpidly metabolised by one of two routes, (1) condensation with serineto form cystathione or (2) conversion to methionine, and itsconcentration (and that of its oxidised form homocystine) in the livingbody under normal conditions is virtually negligible.

Homocysteine levels in biological samples may however be of clinicalsignificance in a number of situations as homocysteine plays animportant part in the complex set of pathways which make up sulphydrylamino acid metabolism and its accumulation may be indicative of variousdisorders occurring in these pathways, including in particular inbornerrors of metabolism. Thus, for example homocystinuria (an abnormalbuild-up of homocysteine in the urine) is known to be a disorder ofamino acid metabolism resulting from deficiencies in the enzymescystathione β synthetase or methyltetrahydro-folic acidmethyltransferase (which catalyses the methylation of homocysteine tomethionine).

Sulphydryl amino acid metabolism is closely linked to that of folic acidand vitamin B₁₂ (cobalamin), which act as substrates or co-factors inthe various transformations involved. For this reason homocysteineaccumulation has also been proposed as an indicator of malfunction ofcobalamin or folate dependent enzymes, or other disorders or diseasesrelated to cobalamin or folate metabolism.

Moreover since homocysteine conversion to methionine relies on areaction requiring S-methyl tetrahydrofolate as the methyl donor,homocysteine metabolism may also be affected by anti-folate drugs, suchas methotrexate, administered to combat other disorders, notably cancer.Monitoring of homocysteine has therefore also been proposed in themanagement of malignant disease treatment with anti-folate drugs.

More recently, elevated levels of homocysteine in the blood have beencorrelated with the development of atherosclerosis (see Clarke et al.,New Eng. J. Med. 324:1149-1155 (1991)) and even moderate homocysteinemiais now regarded as a risk factor for cardiac and vascular diseases.Measurement of plasma or blood levels of homocysteine is thus also ofimportance in the diagnosis and treatment of vascular disease.

Although immunological methods of determining homocysteine directly arenot available as there is no available antibody to homocysteine, anumber of other methods for determining homocysteine in clinical sampleshave been proposed. These have all involved chromatographic separationsand generally have been based on one of the three following principles:

(1) classical chromatographic amino acid analysis,

(2) reaction of homocysteine in the sample with the enzymeS-adenosyl-L-homocysteine hydrolase in the presence of a radioactivelyor otherwise labelled S-adenosine co-substrate followed by separationand quantitation of the product (S-adenosyl-L-homocysteine, SAH) formed.Generally chromatographic separation (HPLC or TLC) and radioactivitymeasurements are used (see Refsum et al., Clin. Chem. 31:624-628 (1985);Kredich et al., Anal. Biochem 116:503-510 (1981); Chui, Am. J. Clin.Path. 90(4):446-449 (1988); Totani et al., Biochea. Soc. 14(6):1172-9(1988); and Schimizu et al., Biotechnol. Appl. Biochem 8:153-159 (1986))

(3) reaction of homocysteine in the sample with a fluorophore, followedby HPLC-separation and fluorometry (see Refsum et al., Clin. Chem.35(9); 1921-1927 (1989)).

These methods are time-consuming and cumbersome to perform and all relyon direct quantitation. More particularly, chromatographic separation isa common feature of the prior art methods and requires highlyspecialised and sophisticated equipment.

The use of such equipment is generally not well accepted in routineclinical laboratory practice and such processes are consequently notgenerally amenable to automation in typical clinical laboratoryprocedures.

A need therefore exists for an improved assay for homocysteine which issimple, specific, quick to perform, readily adapted for use in clinicallaboratories and above all which avoids the need for costly andtime-consuming chromatographic separation. The present invention seeksto provide such an assay.

In one aspect the present invention therefore provides a method forassaying homocysteine in a sample, said method comprising the steps ofcontacting the sample with a homocysteine converting enzyme, eg. anS-adenosyl homocysteine (SAH) hydrolase, and at least one substrate forsaid enzyme other than homocysteine, and without chromatographicseparation (i.e. of reagents or reaction products) assessing (preferablyphotometrically) a non-labelled analyte selected from the homocysteineco-substrate and the products of the enzymic conversion of homocysteineby said enzyme.

After contacting the sample with the homocysteine converting enzyme andthe substrate, it is preferably incubated for at least 30 seconds,especially at least 5 minutes before the subsequent stages of the assayare performed.

The homocysteine converting enzyme used in the assay of the invention isespecially preferably SAH-hydrolase but other enzymes may be used. Thusmention may be made for example of betaine-homocysteine methyltransferase and other enzymes involved in homocysteine conversions, (asdescribed for example by Graham in Trends Cardiovasc. Med. 1: 244-249(1991)).

The homocysteine co-substrate assessed in the method of the invention isa compound which reacts with homocysteine in the enzyme catalysed, e.g.a SAH-hydrolase catalysed, homocysteine conversion reaction.

As used herein the term "assessing" is intended to include bothquantitative and qualitative determination in the sense of obtaining anabsolute value for the amount or concentration of the analyte, e.g.homocysteine co-substrate, present in the sample, and also obtaining anindex, ratio, percentage, visual or other value indicative of the levelof analyte in the sample. Assessment may be direct or indirect and thechemical species actually detected need not of course be the analyteitself but may for example be a derivative thereof or some furthersubstance as discussed below.

The assay of the invention conveniently uses either enzymic orimmunological techniques for analyte assessment. In one preferredenzymic technique the analyte is contacted with a further enzyme forwhich it is a substrate and either a co-substrate or a direct orindirect reaction product of the enzymic conversion of the analyte bythat further enzyme is assessed. In a preferred immunological techniquethe analyte is assessed using a procedure involving competitive bindingto an antibody by the analyte and a further hapten (e.g. a polyhapten ora labelled analogue of the analyte) and assessment of the bound orunbound hapten.

The preferred homocysteine converting enzyme used according to theinvention is S-adenosyl-homocysteine hydrolase (SAH-hydrolase) whichcatalyses the homocysteine reaction ##STR1## a reaction which has anequilibrium constant K of 10⁶ M⁻¹.

The reaction may run in either direction, depending on reactionconditions, reactant concentration etc.

In the above scheme, adenosine is the homocysteine co-substrate. Otherco-substrates such as adenosine analogues or related compounds howevermay be used in the assay method of the invention.

The assay of the invention can take particular advantage of the factthat homocysteine acts as an inhibitor of SAH-hydrolase, suppressing thehydrolysis reaction which forms homocysteine and adenosine and pushingthe reaction equilibrium in favour of SAH synthesis.

The amount of homocysteine in a sample thus indirectly influences theformation or consumption of the homocysteine co-substrate, e.g.adenosine, by SAH-hydrolase and thereby its resulting concentration inthe reaction mixture. In this invention the resulting concentration, orchange in the concentration of homocysteine co-substrate, e.g.adenosine, in the reaction mixture can be used as an indicator for theinitial concentration of homocysteine in the sample. Thus where theanalyte is the co-substrate the assay of the invention differs fromprior art methods in that, rather than being assessed directly,homocysteine is assessed indirectly by determining the concentration ofits co-substrate in its enzyme catalysed conversion. This has the directadvantage that detection methods which are suited to typical clinicallaboratory procedures but which were not usable in the prior art assaysfor homocysteine, e.g. photometric methods, may be used so making theassay according to the invention particularly suited for routineclinical use.

In the preferred assay method of the invention, the SAH-hydrolasereaction may be used in either direction. Thus if the test sample iscontacted with adenosine and SAH-hydrolase, an amount of adenosine isconsumed which corresponds to the amount of homocysteine consumed, andthe amount of homocysteine in the sample can thus be determined from thealteration in the adenosine concentration. Adenosine analogues and/oradenosine generating compounds may be used in the place of adenosineitself.

In other preferred embodiments of the invention, the opposite directionof reaction may be used. The test sample may be contacted with SAH(generally in excess) and SAH-hydrolase. Homocysteine and adenosine arethen formed from hydrolysis of the SAH. Any homocysteine present in thetest sample will counteract this net reaction, and thus inhibit theformation of adenosine, the amount of which is monitored.

The SAH-hydrolase substrates used in the method of the invention maythus be SAH or adenosine or analogues and precursors thereof.

Many enzymes are involved in the complex series of pathways ofsulphydryl amino acid metabolism and transmethylation reactions in thebody. These pathways and reactions have been well studied and theregulatory roles of the enzymes concerned investigated. The role of onesuch enzyme, SAH-hydrolase, is discussed in a review by Ueland inPharmacological Reviews 34: 223-253 (1982). Trewyn et al., in J.Biochem. Biophys. Met. 4: 299-307 (1981), describe an investigation intothe regulatory role of SAH-hydrolase and provide an assay forSAH-hydrolase enzymatic activity. Garras et al. in Analytical Biochem.199: 112-118 (1991) described other homocysteine reaction pathways andin particular provide an assay for the methionine synthase mediatedconversion of homocysteine. As mentioned earlier, Graham (Supra)describes further enzyme mediated homocysteine conversions. Theco-substrates and the conversion products of these various reactions maybe used as the analytes in the assay of the invention, especially wherean immunological means of assessment is employed.

Clinical samples to be assayed according to the invention may be derivedfrom any biological fluid or tissue extract and may be pretreated priorto assay. Plasma or urine samples will however generally be used.

In the plasma or urine, significant proportions of the homocysteinepresent may be bound by disulphide linkage to circulating proteins, suchas albumin, and homocysteine may also be present in the form of otherdisulphide derivatives (generally homocysteine-cysteine conjugates). Toobtain an estimate of total homocysteine present in the sample it maytherefore be desirable to treat the sample with a reducing agent tocleave the disulphide bonds and liberate free homocysteine.

Disulphides are easily and specifically reduced by thiols (e.g.dithiothreitol (DTT), dithioerythritol (DTE), 2-mercapto-ethanol,cysteine-thioglycolate, thioglycolic acid, glutathione and similarcompounds). Direct chemical reduction can be achieved using borohydrides(e.g. sodium borohydride) or amalgams (e.g. sodium amalgam) or morespecialized reagents such as phosphines or phosphorothioates can beused. Disulphide reduction is reviewed by Jocelyn in Methods ofEnzymology 143: 243-256 (1987) where a wide range of suitable reducingagents is listed.

Adenosine or the other homocysteine co-substrates may be assessed byknown methods. Generally methods relying upon photometric (e.g.colorimetric, spectrophotometric or fluorometric) detection andimmunological methods are preferred as these may particularly readily beadapted for use in clinical laboratories. Methods based on enzymicreaction or reaction with mono- or polyclonal antibodies areparticularly preferred, as these are simple and quick and can berelatively inexpensive to perform. Thus for example the analyte may beassessed by monitoring the reaction with enzymes which convert itdirectly or indirectly to products which may be detectedphotometrically, e.g. spectrophotometrically. Suitable enzymes, whichshould of course be non-reactive with the other substrates of thehomocysteine converting enzyme, particularly homocysteine, includeadenosine deaminase (which converts adenosine to inosine) and adenosinekinase (which converts adenosine and ATP to ADP and phosphorylatedadenosine). Such enzymes may further be combined with other enzymeswhich act to convert the products formed to further detectable products.

Examples of immunological methods would include methods involvingreaction of the analyte with antibodies specific for it which either arethemselves assessable or which can be reacted further to form detectableproducts, eg. in a sandwich assay. One particularly attractiveimmunological method however involves the use of a fluorophore labelledanalogue of the analyte, preferably a co-substrate, eg. fluoresceinlabelled adenosine--this and the unlabelled analyte may be contactedwith an antibody for the analyte. If the resultant product is subjectedto a fluorescence polarization assay using polarised exciting radiationan indication of the unlabelled analyte concentration may then bederived from the degree of depolarization of the fluorescence radiation.Adenosine antibodies are commercially available (eg. from Paessel &Lorei GmbH, Frankfurt, Germany and Serotech Ltd., Oxford, UnitedKingdom) and fluorescence polarization immunoassay (FPIA) techniques arewell established (see for example U.S. Pat. No. 4,420,568 and U.S. Pat.No. 4,593,089 and other publications by Abbott Laboratories relating totheir TDx technology).

Thus examples of detection schemes useful in the assay of the inventioninclude ##STR2## or with a fluorophore labelled adenosine which competesfor the ATP/adenosine kinase, with assessment being performed byfluorescence polarization measurement.

As regards schemes (2) and (3), inosine and uric acid have distinctiveUV absorption properties and can thus be monitoredspectrophotometrically, by kinetic measurements.

However the use of UV detection of uric acid or inosine has certainlimitations in that the sensitivity of the method is rather poor and itrequires a UV-light source and a UV-transparent sample container. It maythus be more convenient to rely upon calorimetric detection orelectronic sensors, and such methods, particularly colorimetry, aregenerally favoured in clinical laboratories.

In this connection the reaction of scheme (2) is particularly useful inthat ammonia generated by the adenosine deaminase reaction may readilybe detected using known calorimetric techniques. Thus for exampleammonia generated in the sample may be reacted to form colouredproducts, the formation of which may be detected spectrophotometrically.One such method, described in Methods of Enzymatic Analysis (Bergmeyer)Volume 1:1049-1056 (1970) relies upon the reaction of ammonia withphenol in the presence of hypochlorite in alkaline conditions to formthe coloured dye indophenol: ##STR3##

Sodium nitroprusside may be used as catalyst. Modifications of themethod using for example various derivatives of phenol may also be used.

The coloured end-product is formed in amounts directly proportional tothe concentration of ammonia, and hence adenosine, in the sample.

In scheme (3), the xanthine oxidase reaction lends itself to detectionusing fluorogens or chromogens, e.g. red-ox indicators, by assessing thereduction/oxidation potential, or by measuring O₂ consumption, or moreparticularly H₂ O₂ formation, for example by the use of electronicsensors. Numerous red-ox indicators can be used for this purpose, and awide range of methods are described in the literature for assaying H₂ O₂and O₂ in solution. Indeed, H₂ O₂ is frequently detected in clinicalassays.

Suitable red-ox indicators include methylene blue, 2,6-dichlorophenol,indophenol and the various red-ox indicators listed in Table 1 of theKodak Laboratory & Research Products, Catalog No. 53, although othersmay of course be used. Enzymes with peroxidase activity, e.g.horseradish peroxidase, may be added to facilitate the red-ox reactions.

If a precipitating chromogen is desired, MTT tetrazolium may be used incombination with xanthine oxidase or other similar enzymes. By the useof a precipitating chromogen or fluorogen, a reading of immobilizedcolour or fluorescence can be obtained to obtain a visual indication ofhomocysteine concentration.

In scheme (4), a chemiluminescent ATP reaction is used to assessadenosine concentration. Chemiluminescence based assays have greatpotential due to the low detection limits achievable and to the relativesimplicity of the necessary instrumentation. Chemiluminescent reactionscan be used to detect analytes such as ATP or H₂ O₂ and one of the mostefficient and best known such reactions is the firefly bioluminescencereaction ##STR4##

Firefly luciferin has a benzothiazole structure but luciferins fromother biological sources are available which have other structures. Foranalytical purposes ATP, luciferin or luciferase could be assayeddirectly using this reaction. Another chemiluminescent reaction whichcould be used where H₂ O₂ is generated, as for example in scheme (3), isthe luminol reaction (luminol is5-amino-2,3-dihydro-phthalazine-1,4-dione) with hydrogen perioxidasecatalyst which results in light emission at 425 nm.

Hydrogen peroxide, from scheme (3) for example, can also be assessedusing the non enzymic chemiluminescent reactions of peroxioxalate andthe acridinium esters, the latter in aqueous solution at neutral pH.

The use of fluorophore labelled adenosine in scheme (4) and assessmentby fluorescence polarization measurement is feasible due to therelatively broad substrate specificity of adenosine kinase. This broadspecificity can moreover be used to compensate for endogenous adenosine(or other adenosine kinease nucleoside substrates) by adding adenosinekinase to the sample as a pretreatment, preferably in combination withthe reducing agent (e.g. DTT).

Such enzymic pretreatment of the sample is desirable as, in many of theembodiments of the invention, the analyte (e.g. adenosine) is alreadypresent in the sample in varying amounts, thus providing a potentialsource of error in the assay. Background analyte content can becompensated for by running the assay on a portion of the sample withoutusing the homocysteine converting enzyme (e.g. SAH-hydrolase); howeversuch a procedure is time consuming and makes the assay more cumbersome.An alternative is pre-treatment of the sample with an agent serving toconvert or remove the endogenous analyte, e.g. an enzyme such asadenosine kinase or adenosine deaminase which removes the backgroundadenosine. As mentioned above, to avoid unnecessarily increasing thetime required for the assay to be run, such treatment of the sample mayconveniently be effected at the time that it is pre-treated with thereducing agent to liberate the homocysteine.

Besides the use of spectrometric or calorimetric techniques for analyteassessment, other photometric techniques may be used. Among the mostuseful techniques that can be used are particle agglutination andimmunoprecipitation techniques. If polyclonal antibodies are used,direct particle agglutination or direct immunoprecipitation may be used,although where SAH-hydrolase is used as the homocysteine convertingenzyme this will generally not be preferred. However precipitationinhibition or particle agglutination inhibition techniques can be used.These rely on the use of antibody/hapten combinations which onconjugation lead to precipitation or particle aggregation which can bedetected by turbidimetric or nephelometric measurement. Where theantibody/hapten complex formation is inhibited by the analyte, e.g. SAH,the SAH content may be assessed from the reduction inprecipitation/aggregation. The reaction can be expressed as follows##STR5##

Where the assay of the invention is effected using SAH-hydrolase as thehomocysteine converting enzyme, it is advantageous either to store theSAH-hydrolase in the presence of a reducing agent or to treat it with areducing agent prior to its use in the assay. It has been found thatstorage otherwise causes SAH-hydrolase to become inactivated and thisuse of a reducing agent either prevents inactivation during storage orcauses reactivation prior to use.

Various reducing reagents can be used (for example DTT, cysteine,mercaptoethanol, dithioerythritol, sodium borohydride, etc.), howeverDTT is particularly suitable, e.g. at about 5 mM concentration. DTTshould itself be stored at low pH and thus the assay kit canconveniently include a solution of DTT at a low pH (e.g. about 3) butwith a low buffer capacity and a separate solution of SAH-hydrolase,which may be partially or totally inactive, at substantially neutral pHand preferably buffered. When these solutions are combined, the enzymeis reactivated at neutral pH. This combination can if desired take placein the presence of the test sample, or with the test sample addedshortly thereafter, so that the homocysteine liberation is effectedsimultaneously. The other reducing agents mentioned above may similarlybe used for both SAH-hydrolase stabilization/activation and for reducingthe sample to liberate homocysteine.

The use of reducing agents to reactivate inactivated SAH-hydrolase formsa further aspect of the invention. Moreover in a still further aspectthe invention also provides a kit comprising in a first compartment aninactive SAH-hydrolase and in a second compartment a reducing agent,e.g. DTT in an acid medium (for example pH 3). Using this kit theSAH-hydrolase can be mixed with the reducing agent, and so reactivated,immediately prior to its use in the assay.

Other additives may advantageously be used to enhance SAH-hydrolasestability during storage or in the assay itself. These include NAD⁺,glutathione, polyhydric alcohols and sugars (e.g. inositol, sorbitol,xylitol, eryrthritol, glycerol, ethylene glycol, sucrose, lactitol,etc.), soluble polymers such as certain dextrans, and proteins (e.g.carrier proteins).

Where the assay of the invention uses antibodies, these may bepolyclonal but preferably are monoclonal. Where the desired antibodiesare not already commercially available, they may be produced by standardtechniques. Antibodies can thus be raised in animals or hybridomas,either monoclonal or polyclonal, e.g. as described by James Gooding in"Monoclonal antibodies, principle and practice", Academic Press, London,1983, Chapter 3. Monoclones must be sorted to select clones whichdiscriminate between the desired hapten and other substrates for theenzyme(s), e.g. which discriminate between adenosine and SAH. Polyclonalantibodies reactive only with the analyte (e.g. SAH) should be purifiedto remove cross-reacting antibodies, i.e. those reactive with othersubstrates besides the analyte, e.g. with both adenosine and SAH. Thiscan be done by affinity chromatography, e.g. where SAH is the analyte byusing immobilized adenosine.

In the production of the antibody one uses as a hapten either theanalyte itself or another molecule which includes the portion of theanalyte that is considered to be the most appropriate binding region,e.g. a region remote from those regions participating in the enzymicreaction. The hapten is conveniently conjugated to a macromolecule suchas BSA or hemocyanin. For SAH, the desired epitope is preferably at orabout the thioether bridge and thus while one can use SAH itself##STR6## conjugated to a macromolecule, it is preferred to use a"simplified" molecule such as one of formula (I) ##STR7## (where R₁ andR₂ which may be the same or different are hydrogen atoms or OR₄ groups(wherein R₄ is a lower (e.g. C₁₋₆, especially C₁₋₄) aliphatic group suchas an alkyl group, preferably a methyl or ethyl group) or R₁ and R₂together represent an oxygen atom, and R₃ is an amine or carboxylmoiety) or a salt or ester (e.g. with a C₁₋₄ alkanol) thereof, againcoupled to a macromolecule.

Examples of compounds of formula I that may be used as haptens thusinclude ##STR8##

Such "simplified" structures may also be adopted for the labelledanalogues mentioned above used in assays where the analyte and labelledanalogue are involved in a competitive binding reaction with theantibody. Thus the signal giving moiety R.sup.•, which may be selectedfrom fluorophores, chromophores, radiolabels, enzymic, chemiluminescentand other lables conventionally used in immunoassays can be conjugatedto the analyte,

as for example R.sup.• -SAH,

or to a simplified, epitope containing molecule as for example ##STR9##

Such labelled moieties can of course be coupled to particles, polymers,proteins or other materials if desired.

The labelled furanose 6-thioethers and the compounds of formula I arenovel and form further aspects of the invention.

Viewed from a further aspect the invention provides a process for thepreparation of a compound of formula I, said process comprising at leastone of the following steps:

(a) reacting a compound of formula II ##STR10## (wherein R₁ and R₂ areas defined above and each R₅ is a protected hydroxyl group or both R₅together are an alkylenedioxy group (i.e. a protected bis hydroxy group,

such as --OC(CH₃)₂ O--) with a propyl halide of formula III

    R.sub.6 (CH.sub.2).sub.3 Hal                               (III)

(where R₆ is an R₃ group or a protected R₃ group and Hal is a halogenatom, e.g. a bromine atom) followed by removal of any protecting groupsif desired;

(b) (to produce a compound of formula I wherein R₃ is an amino group)reacting a compound of formula II with acrylonitrile and reducing anddeprotecting the cyano propyl thio-ether product obtained; and

(c) esterifying a compound of formula I wherein R₃ is a carboxyl group.

The starting products of formula III can be produced by standardtechniques or are known from the literature. The starting products offormula II may be produced from the corresponding 1-hydroxymethylfuranoses by cis-hydroxy group protection, bromination and subsequentreaction with thiourea and hydrolysis. The 1-hydroxymethyl-furanoses maybe cis-hydroxy group protected by reaction with conventional hydroxyprotecting agents, e.g. acetone.

Examples of reaction schemes for the preparation of compounds of formulaI include the following (compounds (1) and (3) are commerciallyavailable) ##STR11##

UV absorption, absorption of visible light, or fluorescence bysubstances in the reaction mixture, optionally precipitated or otherwiseseparated from the reaction mixture, may be measured at the reaction endpoint (when the signal is stable) or at one or more fixed time points oralternatively a kinetic measurement may be adopted in which severalmeasurements are made at different points of time.

To perform the assay of the invention, the necessary reagents may beadded to the reaction mixture in a sequential manner or simultaneously.However, in many preferred embodiments, one or more of the reactions mayadvantageously be run for some time before the addition of reagents forthe subsequent reaction(s). As an example, where the test sample isreacted with adenosine and SAH hydrolase, the formation of SAH fromhomocysteine and adenosine is preferably allowed to take place for sometime before the adenosine assessment procedure is initiated.

In clinical chemistry analysis, the use of standard curves forcalibration purposes is standard practice. Thus, in the performance ofthe method of this invention, samples of known homocysteine content maybe used in the place of clinical samples to construct a standard curvefor the response/signal to be measured and the homocysteine content ofthe unknown samples may then be calculated by interpolation from thestandard curve. Thus an exact quantification of the signal formingmolecules or the red-ox potentials is not necessary.

The assay method of the present invention may be used for the diagnosisand monitoring of pathological or potentially pathological conditionswhich are related to or manifested in the homocysteine content of bodyfluids or tissues. These include atherosclerosis, blood diseases,vitamin deficiencies and/or inborn errors of metabolism. It may also beused for the evaluation of the effects of pharmaceuticals, such asanti-folate drugs.

In another aspect the invention provides an analytical product,optionally in kit format, for use in the assay of homocysteine in asample, said product comprising: a homocysteine converting enzyme; asubstrate for said enzyme other than homocysteine; a signal formingagent; and, optionally, means for signal assessment.

In one preferred embodiment, the analytical product comprises: ahomocysteine converting enzyme, eg. S-adenosyl-homocysteine hydrolase;one or more substrates for said enzyme other than homocysteine; meansfor generating a detectable derivative of an analyte selected from thehomocysteine co-substrate and the products of the enzymic conversion ofhomocysteine; and, optionally, means for spectrometrically orcolorimetrically assessing said detectable derivative to provide anindication of the homocysteine content of the sample.

In another embodiment, the product comprises: adenosine;S-adenosyl-homocysteine hydrolase; an adenosine converting enzyme;optionally, a co-substrate for said adenosine converting enzyme; andmeans for generating a photometrically detectable response from saidco-substrate or from a product of enzymic conversion of adenosine bysaid adenosine converting enzyme. Thus for example the adenosineconverting enzyme may be adenosine kinase and the means for generatingmay comprise ATP, luciferin and a luciferase. Alternatively, theadenosine converting enzyme may be adenosine deaminase and the means forconverting may comprise nucleoside phosphorylase, xanthine oxidase and aperoxidase.

In a still further embodiment the kit comprises adenosine;S-adenosyl-homocysteine; an optionally matrix particle boundanti-S-adenosyl-homocysteine antibody; a polyhapten for said antibody;and, optionally, means for photometrically assessing agglutination orprecipitation of antibody:polyhapten complexes. In this embodiment, thepolyhapten may conveniently be provided by a backbone polymer to whichare conjugated a plurality of furanose 6-thioethers, e.g. leavingpendent residues of formula ##STR12##

These may be produced for example by reacting a carboxylic acid offormula I (or an anhydride or acid halide thereof) with a polymer havinga plurality of pendant amines (e.g. polylysine) or an amine of formula Iwith a polymer having pendent carboxyl groups.

In the kits, all or some of the reagents may be present in dry form, asmay the format/matrix for processing the reactions of the reactionmixture. Similarly the kit may, as indicated, include, as means forassessing a detectable analyte or derivative, relatively inexpensivespectrometric or colorimetric apparatus, e.g. a light source anddetector arrangement preset to detect light intensity at a wavelengthcharacteristic of the detectable analyte, etc. or even a simplecalorimetric calibration chart.

The invention will now be described by means of the followingnon-limiting Examples. The assay of Example 19 is especially preferred.

EXAMPLE 1

The sample (an aqueous homocysteine solution for calibration or plasmaor urine for clinical assay) was pretreated with a reducing agent (e.g.10 mM dithiothreitol). This sample was added, preferably to a finalconcentration of homocysteine in the range 10⁻⁶ -10⁻⁵ mol/l, to asolution at 37° C. comprising rabbit IgG 5 mg/ml, adenosine deaminase 20mU/ml, nucleoside phosphorylase 20 mU/ml, xanthine oxidase 20 mU/ml,horseradish peroxidase 500 mU/ml, 10 mmol/l dithiothreitol, 100 mmol/lsodium phosphate, pH adjusted to 7.40, and 100 mUS-adenosyl-l-homocysteine hydrolase. Either simultaneously orsubsequently, but preferably after 10 minutes incubation to allowconversion of adenosine within the sample, adenosine was added to afinal concentration of 5.10⁻⁶ mol/l. UV absorption was measured during a10 minute period and the response was measured at 292 nm in a kineticmode, and the ΔA/Δt was calculated.

For clinical tests the homocysteine concentration may be calculated byinterpolation into a standard curve produced using the known standards.

EXAMPLE 2

The sample (as for Example 1) was pretreated with a reducing agent (e.g.10 mM dithiothreitol). This sample was added, preferably to a finalconcentration of homocysteine in the range 10⁻⁶ -10⁻⁵ mol/l, to asolution at 37° C. comprising rabbit IgG 5 mg/ml, adenosine deaminase 20mU/ml, xanthine oxidase 20 mU/ml, nucleoside phosphorylase 20 mU/ml,horseradish peroxidase 500 mU/ml, 10 mmol/l dithiothreitol and 100mmol/l sodium phosphate, pH adjusted to 7.40, 20 mUS-adenosyl-l-homocysteine hydrolase. Then, preferably after 10 minutesincubation to allow conversion of adenosine within the sample to occur,S-adenosyl-l-homocysteine was added to a final concentration of 5.10⁻⁵mol/l, and the UV absorption was measured during a 10 minute period. At292 nm in a kinetic mode, the ΔA/Δt was calculated.

For clinical tests the homocysteine concentration may be calculated byinterpolation into a standard curve produced using the known standards.

EXAMPLE 3

Assay Buffer:

0.1M phosphate buffer pH 7.4, comprising 1 mg/ml rabbit IgG and 10mmol/l dithiothreitol.

Assay Procedure:

To 150 μl assay buffer, 3 mU S-adenosyl-l-homocysteine hydrolase andsample (preferably pretreated as described in Examples 1 and 2) wereadded. The enzyme reaction was started by addition of adenosinedissolved in assay buffer, to a final concentration of 2.5×10⁻⁵ mol/l.After 10 minutes incubation, 750 μl of a solution of assay buffercomprising 20 mU adenosine deaminase, 20 mU nucleoside phosphorylase, 20mU xanthine oxidase and 375 mU of horseradish peroxidase were added. TheUV absorption at 292 nm was measured for 5 minutes in a kinetic mode.The ΔA/Δt is calculated. In parallel, the assay is repeated but withoutthe addition of S-adenosyl-l-homocysteine hydrolase. The differencebetween the two values of ΔA/Δt is determined and the homocysteineconcentration is calculated by interpolation into a standard curve.

EXAMPLE 4

The assay procedure is performed as described in Example 3 up to thefirst incubation. Then, after 10 minutes incubation, an assay solutioncomprising fluorescein-labelled adenosine and monoclonal anti-adenosineantibodies is added. The remaining adenosine and the fluoresceinlabelled adenosine compete for binding to the antibody. The amount oflabelled adenosine bound to the antibodies is assessed by theconventional fluorescence polarisation technique, and the homocysteineconcentration is calculated by interpolation into a standard curve.

EXAMPLE 5

Assay Buffer:

0.1M phosphate buffer pH 7.4, comprising 1 mg/ml rabbit IgG and 10mmol/l dithiothreitol.

SAH-hydrolase Solution:

40 mU/ml S-adenosyl-l-homocysteine-hydrolase are dissolved in the assaybuffer.

Adenosine Solution:

5×10⁻⁸ mol/ml adenosine are dissolved in the assay buffer.

Adenosine Deaminase Solution:

200 mU/ml adenosine deaminase dissolved in the assay buffer.

Phenol/Nitroprusside Solution:

10 mg/ml phenol and 50 μg/ml sodium nitroprusside in water.

Hypochlorite Solution:

11 mmol/l NaOCl is dissolved in 125 mM NaOH.

Assay Procedure:

1. 75 μl of the adenosine solution and 75 μl of the SAH-hydrolasesolution are mixed with the sample (preferably pretreated as describedin Examples 1 to 4), and kept at 37° C. for 10 minutes.

2. 100 μl of adenosine deaminase solution is added, and the mixture iskept at 37° C. for 5 minutes.

3. 750 μl of phenol/nitroprusside solution and 750 μl hypochloritesolution is added. After 30 minutes in 37° C., the extinction at 628 nmis measured. In parallel the assay is repeated but without the additionof SAH-hydrolase. The difference between the two values for extinctionat 628 nm is determined and the homocysteine concentration is calculatedby interpolation of this difference into a standard curve produced usingknown standards.

EXAMPLE 6

FORMATION OF FLUOROPHORE-LABELLED SAH

A 10 mmol/l solution of SAH in dimethylformamide is prepared and thendiluted 1:10 in a 100 mmol/l phosphate buffer pH=7.5. To this solutionfluorescein isothiocyanate is added to a final concentration of 1mmol/l. After 60 minutes incubation at ambient temperature, theSAH-fluorescein conjugate is purified by HPLC using a Chromasil C-18column at 260 nm using a gradient mixture of 25 mM ammonium acetate (pH7.0) and methanol.

EXAMPLE 7 FORMATION OF ANTI-SAH-ANTIBODIES

a) Formation of Antigen:

To a 1 mmol/l solution of SAH in 25 mmol/l phoshpate buffer pH=7.4, with125 mmol/l NaCl, bovine serum albumin is added to a final concentrationof 5 mg/ml. To this mixture, bis(sulfosuccinimidyl)suberate is added toa final concentration of 1 mmol/l, and the mixture is left to react for60 minutes. (The pH is kept low in this conjugation reaction tostimulate conjugation at the adenosyl amine rather than at thehomocysteine amine function). The proteinaceous fraction of thesolution--which also comprises the conjugates between BSA and SAH--isisolated by size exclusion chromatography using a Pharmacia Superose 12column with phosphate buffered saline as eluant.

b) Formation of Hybridomas:

With the antigen described, hybridomas are formed according to theprocedure described by James W. Gooding in "Monoclonal antibodies:Principle and Practice", Academic Press, London, 1983, Chapter 3.

c) Selection of Hybridomas:

(i) Hybridomas producing anti-SAH-antibodies are identified as follows:The IgG content of the supernate of the hybridomas is measured byconventional ELISA technique. Then supernate is mixed in a cuvette witha buffer comprising 50 mmol/l phosphate, 120 mmol/l NaCl, pH=7.4, and0.1 mg rabbit IgG per ml, to a final concentration of 0.1 μmol/l mouseIgG. Fluorescein labelled SAH, formed according to Example 6 above, isadded to a final concentration of 0.02 μmol/l. After 10 minutesincubation, the degree of polarization is measured and with aspectrofluorometer equipped with a fluorescence polarization unit bymeasuring

A=the fluorescence intensity when the plane of polarization of incidentlight is parallel to the polarization plane of the filter used forfiltration of the emitted light,

B=the fluorescence intensity when the plane of the polarization ofincident light is perpendicular to the polarization plane of the filterused for filtration of the emitted light,

and using an excitation wavelength of 494 nm and detecting the emittedlight at 517 nm.

The degree of polarization is calculated as

    (A-B)/(A+B)

A low degree of polarization indicates that the monoclonal mouseantibodies do not bind SAH.

(ii) From the hybridomas selected according to (i), the hybridomasproducing antibodies reactive to adenosine and/or homocysteine areidentified as follows: Monoclonal anti-SAH-antibodies from the hybridomasupernate is coated onto microtitre well surfaces as described inExample 9 below. Carbon-14 labelled adenosine (or carbon-14 labelledhomocysteine), available from Amersham Ltd, UK, in a buffer comprising25 mmol/l phosphate, 120 mmol/l NaCl and 1 mg/ml of rabbig IgG andhaving pH=7.4 is added to the microtitre wells. After 60 minutesincubation, the wells are washed 3 times with the same buffer (not ofcourse containing the adenosine or homocysteine). High values ofradioactivity retained in the wells indicate that the hybridomas produceantibodies which bind to adenosine (or homocysteine) on its own. Theseantibodies are not used in the assay.

(iii) Production of monoclonal IgG: Hybridomas which produce antibodieswhich bind to SAH according to (i) above but which do not bind toadenosine or homocysteine according to (ii) above are selected formonoclonal IgG production. The hybridomas selected are used forproduction of ascites in mice or production of cell cultures in vitro,according to conventional techniques. The monoclonal antibodies arefurthermore isolated from the ascites or cell culture media according toconventional techniques. See James W. Gooding "Monoclonal antibodies:Principle and practice", Academic Press, London, 1983.

EXAMPLE 8 FLUORESCENCE POLARIZATION IMMUNOASSAY OF L-HOMOCYSTEINE

Enzyme Solution:

50 mmol/l phosphate buffer pH=7.4 comprising 0.2 mg/ml rabbit IgG, 120mmol/l NaCl, 10 mmol/l dithiothreitol, 10 U/lS-adenosyl-l-homocysteine-hydrolase and 0.1 mmol/l adenosine.

Fluorescein-Labelled SAH Solution:

SAH conjugated with fluorescein, formed according to Example 6, isdissolved to a final concentration of 1 μmol/l in 50 mmol/l phosphatebuffer pH=7.4 with 125 mmol/l NaCl and 0.2 mg/ml rabbit IgG.

Antibody Solution:

Monoclonal anti-SAH antibodies (not reactive with adenosine andpreferably also not reactive with homocysteine), e.g. formed accordingto Example 7, dissolved to a final concentration of 0.1 μmol/l in 50mmol/l phosphate buffer pH=7.4 with 125 mmol/l NaCl and 0.2 mg/ml rabbitIgG.

Assay Performance:

In a cuvette, 15 μl plasma (initially a series of samples of knownhomocysteine content) is mixed with 100 μl enzyme solution and kept at37 degrees Celsius for 15 minutes. 100 μl of the solution offluorescein-labelled SAH is added, followed by the addition of 1.0 ml ofthe antibody solution. With a spectrofluorometer equipped with afluorescence polarization unit, the degree of polarization is measuredas described in Example 7 (c) (i) above and is plotted against thehomocysteine concentration.

The assay can also be performed using the labelled haptens andantibodies of Examples 11 and 13 or 15 and 17 in place of those ofExamples 6 and 7.

EXAMPLE 9 MICROTITRE ENZYME-LINKED IMMUNOASSAY

(a) The Enzyme solution of Example 8 is used.

(b) Solution of peroxidase-labelled SAH:

0.5 mg horseradish peroxidase is dissolved in 1 ml purified water. 200μl of a solution of 0.02 mol/l sodium periodate is added, the mixture isstirred for 20 minutes at ambient temperature, and dialyzed overnightagainst a 10 mM sodium acetate buffer pH=4.4. SAH is added to a finalconcentration of 0.1 mmol/l and the pH is adjusted to 6.0. The solutionis stirred for 4 hours at ambient temperature. 100 μl of freshlyprepared 4 mg/ml aqueous solution of sodium borohydride is added, andthe solution is incubated at 4 degrees Celsius for 2 hours. Theperoxidase and its SAH conjugates are isolated by size exclusionchromatography in a column of Superose 6 (Pharmacia, Sweden).

(c) Anti-SAH-Antibodies Coated on Microtitre Wells:

Polyclonal sheep IgG, from sheep immunized to mouse IgG, is dissolved toa final concentration of 1 mg/ml in 100 mmol/l borate buffer pH=9.0. 300μl of this solution is filled in each of the wells of polystyrenemicrotitre plates. After 120 minutes incubation at 37 degrees Celsius,the wells are washed 5 times with phosphate buffered saline. Thereafter,monoclonal mouse IgG anti-SAH-antibodies formed according to Example 7above, are dissolved in phosphate buffered saline to a finalconcentration of 50 μg/ml. 200 μl of this monoclonal IgG solution isadded to each well and incubated for 120 minutes at 37 degrees Celsius.The wells are then washed 5 times with phosphate buffered salinesolution containing 0.1 mg/ml of rabbig IgG.

(d) Assay Performance

A 25 μl plasma sample (initially a series of samples of knownhomocysteine concentration) is mixed with 500 μl of the enzyme solutionand kept at 37 degrees Celsius for 15 minutes. 50 μl of theperoxidase-labelled SAH solution is added, and, following mixing, 250 μlof this mixture is added to a well in the anti-SAH-antibody coatedmicrotitre wells produced according to (c) above all buffered to pH 7.4.After 60 minutes incubation at 37 degrees Celsius, the wells are washedthree times with phosphate buffered saline containing 0.1 mg/ml rabbitIgG. 100 μl of a 1 mg/ml solution of ortho-phenylenediamine in a 0.1mol/l citrate buffer pH=6.0 containing 0.015% hydrogen peroxide is addedto each well. After 10-30 minutes the light absorbance of each well isread at 450 nm. The absorbance is plotted against the homocysteineconcentration.

EXAMPLE 10 Hasten Production

3-S-(1-Anhydro-D-ribofuranosyl)-thioipropyl amine ##STR13## (a)Activated Hydroxy-Protected mercaptan (Compound (8) in Scheme (E) above)##STR14##

One equivalent of methyl β-D-ribofuranoside (Compound (1) above which iscommercially available) is reacted with a mixture of 5 equivalents oftrimethylsilylmethane sulphonate and one equivalent of boron trifluorideetherate using the procedure of Jun. et al. (Carb. Res. 163: 247-261(1987)). 0.5 equivalents of the 1-anhydro-D-ribose product (Compound(2)) is added in finely powdered form, in small portions, undercontinuous stirring to an acetone/sulphuric acid mixture produced byadding 6.3 ml of concentrated sulphuric acid slowly to 100 ml of freshlydistilled acetone in an ice bath. The ice bath is removed and reactionis allowed to continue at ambient temperature for 8 hours. The solidwhite crystalline mass obtained is dissolved in chloroform, washed withaqueous sodium hydroxide, dilute hydrochloric acid and finally water,dried and evaporated down to yield the 2,3-isopropylidene-D-ribonicderivative of 1-anhydro-D-ribose (Compound (2)). One equivalent of thisand two equivalents of carbon tetrabromide are dissolved in dry etherand cooled on ice. With constant stirring and cooling on ice, twoequivalents of triphenylphosphine are added slowly. The ice bath isremoved and the mixture is allowed to warm up to ambient temperatureletting the reaction take place and causing hydrogen bromide to evolvesmoothly. After the reaction is complete, excess reagent is quenchedwith the addition of methanol. The bromide derivative (Compound (6)) isisolated by filtration and evaporation down of the filtrate. To thebromide derivative is added thiourea (one equivalent) dissolved in warmwater and diluted with rectified spirit. The mixture is refluxed andshaken well periodically, this continuing until about 30 minutes afterthe bromide derivative dissolves. The reaction mixture is cooled on iceand filtered to yield a solid which is treated with alkaline waterproducing the hydroxy-protected mercaptan (Compound (7)) in the organicphase. This is converted to the activated form (Compound (8)) bytreatment with methoxide in methanol. This is then reacted further asdescribed below.

(b) 3-S-(1-Anhydro-D-ribofuranosyl)thiopropylamine

One equivalent of the compound of Example 10(a) (Compound (8)) freshlyprepared is reacted with one equivalent of acrylonitrile to yield athioether (Compound (9)). This is then reduced by treatment with LiAlH₄in dry ether and the free deprotected amine (Compound (10)) is isolatedby treatment with aqueous hydrochloric acid.

The corresponding aminopropylthioethers in which R₁ and R₂ are otherthan hydrogen are produced analogously, e.g. using the commerciallyavailable compounds (1) and (3) as starting materials.

EXAMPLE 11 Hapten Labelling

A 50 mmol/l solution of the compound of Example 10 in dimethylformamide(DMF) is diluted 1:5 (by volume) in 0.1M bicarbonate solution (pH 9.2).To this solution, fluorescein isothiocyanate is added to a finalconcentration of 12 mmol/l. After 60 minutes incubation at ambienttemperature, the fluorescein conjugate of the compound of Example 10 ispurified by RPC using a Kromasil 100 Å C-18 column and a gradientmixture of 20 mM ammonium acetate (pH 7.0) and methanol.

EXAMPLE 12 Antigen Preparation

To a 1 mmol/l solution of the compound of Example 10 in 50 mmol/lphosphate, 125 mmol/l NaCl (pH 7.4) buffer, bovine serum albumin (BSA)is added to a final concentration of 5 mg/ml. To this mixture,bis(sulfosuccinimidyl)suberate is added to a final concentration of 1.2mmol/l and the mixture is left to react for 60 minutes. Theproteinaceous fraction, which includes the hapten-BSA conjugate, isisolated by size exclusion chromatography using a Pharmacia Superose 12column with phosphate buffered saline as eluant.

EXAMPLE 13 Antibody Preparation

Antibodies to the compound of Example 10 are prepared analogously toExample 7 above using the antigen of Example 12. Antibodies not reactivewith SAH and antibodies reactive with adenosine are rejected aspreferably are antibodies reactive with homocysteine.

EXAMPLE 14 Hapten Production

N-Hydroxy succinimidyl 3-S-(1-Anhydro-D-ribofuranosyl)thiobutanoate##STR15##

One equivalent of the compound of Example 10(a), freshly prepared, isreacted with 1 equivalent of ethyl 4-bromo-butyrate to yield the estercompound (11). This is hydrolysed to the free acid by basic hydrolysiswith aqueous sodium hydroxide in dioxane and deprotected by treatmentwith aqueous hydrochloric acid to yield the unprotected free acid(Compound (12)). One equivalent of this is mixed with two equivalents ofN-hydroxysuccinimide in ice-cold dimethylformamide and to this is added1.2 equivalents of dicyclohexylcarbodiimide under constant stirring. Thereaction is allowed to proceed at ambient temperature for 18 hours,whereafter the mixture is cooled and ice-cold ether is added. Theprecipitated NHS-ester (Compound (13)) is recrystallized from DMF/ether,dried and then stored at 4° C. over a desiccant.

The corresponding NHS-esters in which R₁ and R₂ are other than hydrogenare produced analogously, e.g. using the commercially availablecompounds (1) and (3) as starting materials.

EXAMPLE 15 Hasten Labelling

A 50 mmol/l solution of the compound of Example 14 in DMF is diluted 1:5(by volume) in 0.1 M bicarbonate buffer (pH 9.2). To this solution,5-aminoacetamido-fluorescein (fluoresceinyl glycine amide) as added to afinal concentration of 12 mmol/l. After 60 minutes incubation at ambienttemperature, the fluorescein conjugate of3-S-(1-anhydro-D-ribofuranosyl)thio-butanoic acid is purified by RPCusing a Kromasil 100 Å, C-18 column and a gradient mixture of 20 mMammonium acetate (pH 7.0) and methanol.

EXAMPLE 16 Antigen Preparation

To a solution of BSA (5 mg/l in 50 mmol/l phosphate, 125 mmol/l NaCl (pH7.4) buffer), the compound of Example 14 is added to a finalconcentration of 1 mmol/l. The mixture is left to react for 60 minutesand the proteinaceous fraction, which contains the BSA-hapten conjugate,is isolated by size exclusion chromatogrpahy using a Pharmacia Superose12 column with phosphate buffered saline as eluant.

EXAMPLE 17 Antibody Preparation

Antibodies to the compound of Example 14 are prepared analogously toExample 7 above using the antigen of Example 12. Antibodies not reactivewith SAH and antibodies reactive with adenosine are rejected aspreferably are antibodies reactive with homocysteine.

EXAMPLE 18 Fluorescence Polarization Immunoassay

Enzyme Solution:

50 mmol/l phosphate buffer (pH 7.4) containing 4 mg/ml casein, 120mmol/l NaCl, and 10 U/1 S-adenosyl-L-homocysteine-hydrolase.

Dithiothreitol (DTT)-Solution:

Dithiothreitol is dissolved in water to a concentration of 50 mmol/l andadjusted to pH 3.0 with HCl.

Adenosine-Solution:

1.8 mmol/l adenosine in 50 mmol/l phosphate buffer (pH 7.4).

Fluorescein-Labelled SAH Solution:

50 mmol/l phosphate buffer (pH 7.4) containing SAH conjugated tofluorescein, formed according to Example 6.

Antibody Solution:

Monoclonal anti-SAH antibodies (e.g. according to Example 7) dissolvedto a final concentration of 0.1 μmol/l in 50 mmol/l phosphate buffer (pH7.4) containing 120 mmol/l NaCl and 1 mg/ml casein.

Assay Performance

Step 1:

In a cuvette, 15 μl of sample, 10 μl enzyme solution and 10 μl adenosinesolution are mixed with 10 μl of the acidic DTT-solution and kept at 37°C. for 15 minutes.

Step 2:

To the cuvette, 100 μl of the fluorescein-labelled SAH solution and 1.0ml of the antibody solution are added.

With a spectrofluorometer equipped with a fluorescence polarizationunit, the degree of polarization is measured as described in Example 7(c) (i) above and is plotted against the homocysteine concentration.

EXAMPLE 19 Fluorescence Polarization Immunoassay

Enzyme Solution:

50 mmol/l phosphate buffer (pH 7.4) containing 1 mg/ml casein, 120mmol/l NaCl, and 10 U/l S-adenosyl-L-homocysteine-hydrolase.

Dithiothreitol (DTT)-Solution:

Dithiothreitol is dissolved in water to a concentration of 50 mmol/l andadjusted to pH 3.0 with HCl.

Fluorescein-Labelled SAH Solution/Adenosine-Solution:

50 mmol/l phosphate buffer (pH 7.4) containing 10 μmol/l SAH conjugatedto fluorescein, formed according to Example 6, and 1.8 mmol/l adenosine.

Antibody Solution:

Monoclonal anti-SAH antibodies (e.g. according to Example 7) dissolvedto a final concentration of 0.1 μmol/l in 50 mmol/l phosphate buffer (pH7.4) containing 120 mmol/l NaCl and 1 mg/ml casein.

Assay Performance

In a cuvette, 10 μl plasma, 100 μl enzyme solution and 10 μl labelledSAH/adenosine-solution are mixed with 30 μl of the acidic DTT-solutionand kept at 37° C. for 15 minutes.

After incubation, 1.0 ml of the antibody solution is added. With aspectrofluorometer equipped with a fluorescence polarization unit, thedegree of polarization is measured as described in Example 7(c) (i)above and is plotted against the homocysteine concentration.

The assays of Examples 18 and 19 can also be performed using thelabelled haptens and antibodies of Examples 11 and 13 or 15 and 17 inplace of those of Examples 6 and 7.

EXAMPLE 20 Luminescence Assay

Assay Buffer I:

50 mM Pipes-buffer (pH 6.6) containing 1 mg/ml casein, 10 mM DTT, 0.5 mMMgCl₂, and 30 mM KCl.

Assay Buffer II:

40 mM Hepes-buffer (pH 7.75), 4 mmol/l EDTA, 20 mM magnesium chlorideand 0.36 mmol/l DTT.

Assay Buffer III:

40 mM Hepes-buffer (pH 7.75) containing 1.6 μg/ml luciferase (fromPhotinus pyralis), 700 μmol/l D-luciferin, 20 mmol/l magnesium chloride,4 mmol/l EDTA, 0.36 mmol/l DTT and 0.3 mmol/l AMP.

Assay Procedure:

To 130 μl of Assay buffer I is added 3 U of S-adenosyl-l-homocysteinehydrolase, 20 μl sample is added to this mixture and it is incubated for15 minutes at 37 degrees Celsius. Thereafter, adenosine dissolved inAssay buffer I to a final concentration of 5×10⁻⁶ mol/l is added. After5 minutes incubation at 37° C., 750 μl of Assay buffer I containing0.7×10⁻⁵ mol/l ATP and 1 mU adenosine kinase are added, and theresulting solution is further incubated at 37° C. for 5 minutes. Thissolution is diluted 1:100 (by volume) with Assay buffer II and 500 μl ofthis diluted solution is immediately added to 500 μl of Assay bufferIII. Both Assay buffers II and III were equilibrated to ambienttemperature (21° C.). The luminescence produced is read in a photometerat 550 nm.

In parallel, an assay with no S-adenosyl-l-homocysteine hydrolasepresent is run. For clinical tests the homocysteine concentrations maybe calculated by interpolation into a standard curve the difference inluminescence produced with and without S-adenosyl-l-homocysteinehydrolase present.

EXAMPLE 21 Polyclonal Antibody Preparation

Rabbit polyclonal antibodies to the antigens of Examples 12 and 16 areraised according to the protocol issued by the Dako Corporation,Copenhagen, Denmark. Polyclonal IgG is purified from the collectedantiserum according to the same protocol. The polyclonal antibodies arepurified from antibodies reactive with adenosine and homocysteineresidues Per se by passing the antibodies through Racti-Gel columns withimmobilized adenosine and homocysteine residues (Gel and protocol asprovided by Pierce Chemical Company, Belfium). Antibodies unreactivewith SAH are also rejected. The selected antibodies can be used in theassays of the earlier Examples.

I claim:
 1. An immunological method for indirectly assaying homocysteinein a sample, said method comprising the steps of:(a) contacting saidsample with S-adenosyl homocysteine hydrolase enzyme and adenosine or ananalogue of adenosine, wherein said S-adenosyl homocysteine hydrolaseconverts said homocysteine into a non-labelled analyte, wherein saidnon-labelled analyte is S-adenosyl homocysteine or an analogue thereof;and (b) determining the presence or amount of the non-labelled analytewithout chromatographic separation by contacting said sample with anantibody which specifically binds with said non-labelled analyte andwith a detectable hapten for said antibody other than said non-labelledanalyte, and wherein determining the presence or amount of saidnon-labelled analyte is effected indirectly by determining the presenceor amount of said detectable hapten either bound or not bound to saidantibody, wherein the amount of the non-labelled analyte is directlyproportional to the amount of homocysteine in said sample.
 2. The methodas claimed in claim 1 wherein said antibody is a monoclonal antibody. 3.The method as claimed in claim 1 wherein said antibody is a carriermatrix bound antibody.
 4. The method as claimed in claim 1 wherein saidsample is a blood, plasma or urine sample pre-treated with a disulphidebond cleaving reducing agent.
 5. The method as claimed in claim 1wherein the determination of the presence or amount of said non-labelledanalyte is effected photometrically.
 6. The method as claimed in claim 5wherein said determination of the presence or amount of saidnon-labelled analyte is effected spectrometrically or calorimetrically.7. The method as claimed in claim 5 wherein said determination of thepresence or amount of said non-labelled analyte is effectedturbidimetrically or nephelometrically.
 8. The method as claimed inclaim 5 wherein said determination of the presence or amount of saidnon-labelled analyte is effected using fluorescence polarizationdetection.
 9. The method as claimed in claim 1 wherein as saiddetectable hapten is a compound of formula I ##STR16## wherein: R₁ andR₂, which may be the same or different denote hydrogen atoms or OR₄groups or R₁ and R₂ together denote an oxygen atom,R₃ denotes an aminoor carboxyl group, and R₄ denotes a C₁₋₆ aliphatic group, or a salt orester thereof.
 10. The method as claimed in claim 1 wherein saiddetectable hapten is a labelled furanose 6-thioether, wherein thelabelled moiety comprises a chromophore or fluorophore or a radioactiveatom, and wherein the thioether moiety comprises a trimethylenethiomoiety.
 11. The method as claimed in claim 10 wherein as said labelledthioether is a labelled compound of formula I: ##STR17## wherein: R₁ andR₂, which may be the same or different denote hydrogen atoms or OR₄groups or R₁ and R₂ together denote an oxygen atom,R₃ denotes an aminoor carboxyl group, and R₄ denotes a C₁₋₆ aliphatic group, or a salt orester thereof.
 12. The method as claimed in claim 1 wherein said enzymewas activated by contact with a reducing agent prior to contacting withsaid sample.
 13. The method as claimed in claim 1 wherein saiddetectable hapten is selected from S-adenosyl homocysteine and afuranose 6-thioether labelled with a chromophore or a fluorophore. 14.The method of claim 1, wherein contacting comprises mixing the samplewith adenosine and S-adenosyl homocysteine-hydrolase to form a firstmixture, incubating the first mixture, contacting the incubated mixturewith a fluorophore labelled compound selected from the group consistingof fluorophore labelled S-adenosyl-homocysteine and fluorophore labelledfuranose 6-thioether to form a second mixture, the second mixture isthen contacted with a monoclonal anti-S-adenoasyl-homocysteine antibody;and wherein determining the presence of the non-labelled analytecomprises detecting the antibody-bound or non-antibody bound fluorophorelabelled compound by fluorescence polarization.
 15. The method of claim1, wherein said contacting comprises mixing the sample with adenosineand S-adenosyl homocysteine-hydrolase to form a first mixture,incubating the first mixture, contacting the incubated mixture with alabelled compound selected from the group consisting of labelledS-adenosyl-homocysteine and fluorophore labelled furanose 6-thioether toform a second mixture, the second mixture is then contacted with acarrier matrix bound monoclonal anti-S-adenosyl-homocysteine antibodyand washing the antibody bound carrier matrix; and wherein determiningthe presence of the non-labelled analyte comprises photometricallydetecting the antibody-bound labelled compound present on the washedcarrier matrix.
 16. The method of claim 15, wherein determining thepresence of the non-labelled analyte further comprises contacting theantibody bound labelled compound on the washed carrier matrix with asubstrate to generate a chromophore which is then photometricallydetected.
 17. An immunological method for indirectly assayinghomocysteine in a sample, said method comprising the steps of:(a)contacting said sample with S-adenosyl homocysteine hydrolase enzyme andS-adenosyl homocysteine wherein said S-adenosyl homocysteine hydrolaseconverts said S-adenosyl-homocysteine into a non-labelled analytewherein said non-labelled analyte is adenosine; and (b) determining thepresence or amount of the non-labelled analyte without chromatographicseparation by contacting said sample with an antibody which specificallybinds with said non-labelled analyte and with a detectable hapten forsaid antibody other than said non-labelled analyte and whereindetermining the presence or amount of said non-labelled analvte iseffected indirectly by determining the presence or amount of saiddetectable hapten either bound or not bound to said antibody, whereinthe amount of the non-labelled analyte is indirectly proportional to theamount of homocysteine in said sample.
 18. The method as claimed inclaim 17 wherein said antibody is a monoclonal antibody.
 19. The methodas claimed in claim 17 wherein said antibody is a carrier matrix boundantibody.
 20. The method as claimed in claim 17 wherein said sample is ablood, plasma, or urine sample pre-treated with a disulphide bondcleaving reducing agent.
 21. The method as claimed in claim 17 whereinthe determination of the presence or amount of said analyte is effectedphotometrically.
 22. The method as claimed in claim 21 wherein saiddetermination of the presence or amount of said analyte is effectedspectrometrically or colorimetrically.
 23. The method as claimed inclaim 21 wherein said determination of the presence or amount of saidanalyte is effected turbidimetrically or nephelometrically.
 24. Themethod as claimed in claim 21 wherein said determination of the presenceor amount of said analyte is effected using fluorescence polarizationdetection.
 25. The method as claimed in claim 17 wherein said enzyme isSAH-hydrolase which has been activated by contact with a reducing agentprior to contacting with said sample.
 26. The method as claimed in claim17, wherein said detectable hapten is adenosine or an analogue thereoflabelled with a chromophore or a fluorophore.
 27. The method as claimedin claim 17 wherein said contacting comprising mixing the sample withS-adenosyl homocysteine and an S-adenosyl homocysteine-hydrolase to forma first mixture; incubating the first mixture, contacting the incubatedmixture with a fluorophore labelled compound selected from the groupconsisting of adenosine or an analogue of adenosine to form a secondmixture; the second mixture is then contacted with a monoclonalanti-adenosine or an antibody against an analogue of adenosine; and theantibody-bound or non-antibody bound fluorophore labelled compound isdetected by fluorescence polarization.
 28. The method as claimed inclaim 17 wherein said contacting comprises mixing the sample withS-adenosyl homocysteine and an S-adenosyl-homocysteine hydrolase to forma first mixture; incubating the first mixture, contacting the incubatedmixture with a labelled adenosine or a labelled analogue of adenosine toform a second mixture; the second mixture is then contacted with carriermatrix bound monoclonal anti-adenosine antibody or an antibody againstan analogue of adenosine; the antibody carrier matrix is washed; and theantibody-bound labelled compound is photometrically detected.
 29. Themethod as claimed in claim 28 wherein the label of the antibody boundlabelled compound is contacted with a substrate to generate achromophore which is then photometrically assessed.